EHD4 and CDH23 Are Interacting Partners in Cochlear Hair Cells

Abstract

Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner. Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner. Hair cells located in the mammalian inner ear transform mechanical stimuli into electrical signals that in turn facilitate neurotransmitter release onto auditory neurons. The key element in the transduction process is the mechano-electric transducer (MET) 2The abbreviations used are: METmechano-electric transducerOHCouter hair cellIHCinner hair cellCDH23cadherin 23PBIPDZ-binding interfaceEHEps 15 homologyKOknock-outWTwild typeEHDEH domain-containing proteinUSH1DUsher syndrome type 1D factorEEearly endosomeERCendocytic recycling compartmentGFPgreen fluorescence proteinHRPhorseradish peroxidaseEHD4ΔEHEHD4 protein without the EH domainOKopossum kidneyHEKhuman embryonic kidneyHSC70heat shock cognate protein 70Co-IPco-immunoprecipitationCAPcompound action potentialNubthe N-terminal domain of ubiquitinNubGthe mutant form of NubCubthe C-terminal domain of ubiquitinBSAbovine serum albuminPBSphosphate-buffered saline. apparatus located near the top of the stereocilium. CDH23 is a single pass transmembrane protein with 27 extracellular cadherin repeats. It is one of the components of the tip-link (1Siemens J. Lillo C. Dumont R.A. Reynolds A. Williams D.S. Gillespie P.G. Müller U. Nature. 2004; 428: 950-955Crossref PubMed Scopus (351) Google Scholar, 2Söllner C. Rauch G.J. Siemens J. Geisler R. Schuster S.C. Müller U. Nicolson T. Nature. 2004; 428: 955-959Crossref PubMed Scopus (267) Google Scholar), which connects the top of a shorter stereocilium to the side of its taller neighbor (3Pickles J.O. Comis S.D. Osborne M.P. Hear Res. 1984; 15: 103-112Crossref PubMed Scopus (525) Google Scholar). Vibrations of the basilar membrane of the inner ear ultimately result in deflection of the hair bundles, which modulates tension on the tip-link, thereby controlling the opening probability of cation-selective MET channels (3Pickles J.O. Comis S.D. Osborne M.P. Hear Res. 1984; 15: 103-112Crossref PubMed Scopus (525) Google Scholar, 4Howard J. Hudspeth A.J. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 3064-3068Crossref PubMed Scopus (300) Google Scholar). Cations, principally K+ and Ca2+, flow through the MET channels and ultimately change the membrane potential. A mutation in the gene encoding CDH23, the Usher syndrome type 1D factor (USH1D), causes deaf-blindness in humans (5El-Amraoui A. Petit C. J. Cell Sci. 2005; 118: 4593-4603Crossref PubMed Scopus (154) Google Scholar). Several interacting partners of CDH23 have been reported and include another tip-link protein protocadherin 15 (6Kazmierczak P. Sakaguchi H. Tokita J. Wilson-Kubalek E.M. Milligan R.A. Müller U. Kachar B. Nature. 2007; 449: 87-91Crossref PubMed Scopus (519) Google Scholar), a multi-PDZ domain-containing scaffold protein harmonin (7Boëda B. El-Amraoui A. Bahloul A. Goodyear R. Daviet L. Blanchard S. Perfettini I. Fath K.R. Shorte S. Reiners J. Houdusse A. Legrain P. Wolfrum U. Richardson G. Petit C. Weil D. EMBO J. 2002; 21: 6689-6699Crossref PubMed Scopus (358) Google Scholar) and a stereociliary scaffolding protein MAGI-1 (8Xu Z. Peng A.W. Oshima K. Heller S. J. Neurosci. 2008; 28: 11269-11276Crossref PubMed Scopus (45) Google Scholar). Protocadherin 15 binds to CDH23 through its extracellular domains (6Kazmierczak P. Sakaguchi H. Tokita J. Wilson-Kubalek E.M. Milligan R.A. Müller U. Kachar B. Nature. 2007; 449: 87-91Crossref PubMed Scopus (519) Google Scholar), whereas the cytoplasmic region of CDH23 interacts with MAGI-1 and harmonin through its PDZ domain-binding interfaces (PBI). Harmonin also associates with other USH1 factors like myosin VIIa, protocadherin 15, and sans (9Adato A. Michel V. Kikkawa Y. Reiners J. Alagramam K.N. Weil D. Yonekawa H. Wolfrum U. El-Amraoui A. Petit C. Hum. Mol. Genet. 2005; 14: 347-356Crossref PubMed Scopus (196) Google Scholar). These findings indicate that harmonin bridges CDH23 to the cytoskeletal actin core of the stereocilium and is probably essential for the developmental differentiation of stereocilia (10Frolenkov G.I. Belyantseva I.A. Friedman T.B. Griffith A.J. Nat. Rev. Genet. 2004; 5: 489-498Crossref PubMed Scopus (173) Google Scholar, 11Michel V. Goodyear R.J. Weil D. Marcotti W. Perfettini I. Wolfrum U. Kros C.J. Richardson G.P. Petit C. Dev. Biol. 2005; 280: 281-294Crossref PubMed Scopus (136) Google Scholar, 12Petit C. Trends Mol. Med. 2006; 12: 57-64Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, it is currently unknown how CDH23 is transported to the tip of stereocilia. To search for additional interacting partners of CDH23, we performed a membrane-based yeast two-hybrid assay, which identified EHD4 as a potential binding partner (13Zheng J. Anderson C.T. Miller K.K. Cheatham M. Dallos P. BMC Genomics. 2009; 10: 127Crossref PubMed Scopus (9) Google Scholar). mechano-electric transducer outer hair cell inner hair cell cadherin 23 PDZ-binding interface Eps 15 homology knock-out wild type EH domain-containing protein Usher syndrome type 1D factor early endosome endocytic recycling compartment green fluorescence protein horseradish peroxidase EHD4 protein without the EH domain opossum kidney human embryonic kidney heat shock cognate protein 70 co-immunoprecipitation compound action potential the N-terminal domain of ubiquitin the mutant form of Nub the C-terminal domain of ubiquitin bovine serum albumin phosphate-buffered saline. EHD4 belongs to an evolutionarily conserved EH (Eps 15 homology) domain-containing protein family involved in endocytic trafficking and recycling. Four highly homologous members of this family, EHD1–4, are expressed in mammalian cells. They contain a single C-terminal EH domain, an N-terminal nucleotide-binding loop and a coiled-coil region responsible for oligomerization (14Lee D.W. Zhao X. Scarselletta S. Schweinsberg P.J. Eisenberg E. Grant B.D. Greene L.E. J. Biol. Chem. 2005; 280: 17213-17220Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15Mintz L. Galperin E. Pasmanik-Chor M. Tulzinsky S. Bromberg Y. Kozak C.A. Joyner A. Fein A. Horowitz M. Genomics. 1999; 59: 66-76Crossref PubMed Scopus (80) Google Scholar, 16Pohl U. Smith J.S. Tachibana I. Ueki K. Lee H.K. Ramaswamy S. Wu Q. Mohrenweiser H.W. Jenkins R.B. Louis D.N. Genomics. 2000; 63: 255-262Crossref PubMed Scopus (83) Google Scholar). Of the four EHD proteins EHD1 is the best characterized and is involved in regulating the recycling of membrane receptors including the transferrin receptor and the major histocompatibility complex class I (17Caplan S. Naslavsky N. Hartnell L.M. Lodge R. Polishchuk R.S. Donaldson J.G. Bonifacino J.S. 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EHD2 was isolated from GLUT4-enriched fractions of adipocytes and was shown to regulate insulin-mediated translocation of GLUT4 to the plasma membrane (23Guilherme A. Soriano N.A. Bose S. Holik J. Bose A. Pomerleau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 24Park S.Y. Ha B.G. Choi G.H. Ryu J. Kim B. Jung C.Y. Lee W. Biochemistry. 2004; PubMed Scopus Google Scholar). EHD2 is involved in the of transferrin receptor (23Guilherme A. Soriano N.A. Bose S. Holik J. Bose A. Pomerleau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), recycling M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar), and actin (23Guilherme A. Soriano N.A. Bose S. Holik J. Bose A. Pomerleau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). EHD4, also was first reported as an extracellular protein J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). have shown this protein to be involved in the of receptor in cells Y. W. J. G. S. J. Cell Biol. 2002; PubMed Scopus Google Scholar). It is also involved in with the cell and co-localizes with the C.A. Donaldson J. U. C.J. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google Scholar). Sharma M. Naslavsky N. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar) showed that EHD4 the of endocytic from the early endosome the recycling compartment and the endocytic They also that EHD4 and EHD1 as of the EHD4 on early EHD1 on recycling be for the of proteins through the early endosome to the M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar) also demonstrated that EHD4 interacts with EHD1 and its which and of EHD4 and other proteins, EHD4 contain a PDZ domain that could to the located in the cytoplasmic of the cytoplasmic of CDH23 an that could an with the EH domain of we to the of EHD4 and CDH23 identified in yeast and mammalian using in and in the expression of EHD4 mRNA in mouse and the role of EHD4 protein in the using mice. encoding and EHD4 and are M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar). R. a extracellular cadherin the transmembrane domain, and the cytoplasmic encoding and were J. W. Dallos P. 2001; 12: PubMed Scopus Google Scholar). were and from from and from EHD1, EHD3, and EHD4 were as M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar), and EHD4 was from was from include and protein and and was into the expression with of and with The the gene for The of the was through of the protein was analysis with of EHD4 was into the with of and with The the gene for is a which the protein in the in the yeast The and the were into yeast and on EHD4 were into yeast or yeast and on were identified the of yeast to on in the of and the in the of and were in with the of and and the of The for cochlear were in B. Res. Res. 2002; PubMed Scopus Google Scholar). wild type and mice were first with and with were and in for A to the EHD4 cDNA was into and from the and were with using the of with the was used to cochlear were on and with a were with a encoding EHD4 or were with that of CDH23 or in opossum kidney cells to the in J. W. Dallos P. 2001; 12: PubMed Scopus Google Scholar). the cells were with in for were in with and for and with or in with and for were in with and normal serum for The were on with and using a with a on the and of mice are M. M. A. G. L. M. H. S. E. V. and H. for a gene knock-out was using the P. Jenkins N.A. Res. PubMed Scopus Google Scholar). cells were used for gene and the first of was the mice with which in a of including the cells. mice used in this have been on the and from wild type or mice were in mammalian with a of and were for to the and The protein was measured using the and bovine serum albumin as a cell was used as an additional lysates were with with and for of of protein were to and with of EHD1, EHD3, EHD4, or of protein were using and cochlear potentials were the membrane using a in mice with A was and the was to of the action potential were for a J. J. Dallos P. J. 2004; PubMed Scopus (129) Google Scholar). were the of and co-immunoprecipitation of proteins, cells were with encoding and with or using cells were and in with a of and were in or in to of the was for 15 were of of protein of and protein Cell with or was used as a were with and proteins were in with The proteins were on a was with and whereas was using a CDH23 protein was with a were using A membrane-based yeast two-hybrid screen was performed to identify potential CDH23 partners from an cDNA library (13Zheng J. Anderson C.T. Miller K.K. Cheatham M. Dallos P. BMC Genomics. 2009; 10: 127Crossref PubMed Scopus (9) Google Scholar). is a highly for protein partners are in the on the cell membrane I. S. Trends Biochem. Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). using CDH23 as the encoding EHD4 were isolated (13Zheng J. Anderson C.T. Miller K.K. Cheatham M. Dallos P. BMC Genomics. 2009; 10: 127Crossref PubMed Scopus (9) Google Scholar). The of these to the EH domain of The and was using to yeast expression of CDH23 and EHD4, as as shown in A and the yeast and on and for the of the gene However, yeast and showed no that EHD4 could with CDH23 but with These data that EHD4 is a potential interacting partner of CDH23 in CDH23 is expressed in hair cells and membrane of the V. L. B. Lee D. Jenkins N.A. Genomics. 2001; PubMed Scopus Google Scholar, A. R.J. Belyantseva I.A. Friedman T.B. Dev. Biol. 2005; 280: PubMed Scopus Google Scholar). To cochlear EHD4 mRNA in hair in situ was performed to EHD4 mRNA the mouse cochlear was with or EHD4 the EHD4 showed signals in and in The which was used as a no no was mouse cochlear was with the EHD4 or the EHD4 These data that EHD4 mRNA is in of receptor cells in mouse CDH23 is to be The of mRNA in hair cells is with the that CDH23 and EHD4 proteins could in the of EHD4 protein in hair cells. However, M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar) and with other EHD (1Siemens J. Lillo C. Dumont R.A. Reynolds A. Williams D.S. Gillespie P.G. Müller U. Nature. 2004; 428: 950-955Crossref PubMed Scopus (351) Google Scholar, 2Söllner C. Rauch G.J. Siemens J. Geisler R. Schuster S.C. Müller U. Nicolson T. Nature. 2004; 428: 955-959Crossref PubMed Scopus (267) Google Scholar, J.O. Comis S.D. Osborne M.P. Hear Res. 1984; 15: 103-112Crossref PubMed Scopus (525) Google Scholar) proteins to homology these it was to EHD4 protein expression in the To investigate the CDH23 and EHD4 proteins, containing and protein without EH were with a into opossum kidney cells. was and CDH23 near the plasma CDH23 is a single domain transmembrane protein whereas EHD4 contain transmembrane However, EHD4 been shown to with through a with or with proteins A. D. M. M. Cell Res. 2007; PubMed Scopus Google Scholar, N. Rahajeng J. S. Caplan S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The of EHD4 with CDH23 in the plasma membrane could a or these It been shown that EHD proteins to of and of the EH domain in in (17Caplan S. Naslavsky N. Hartnell L.M. Lodge R. Polishchuk R.S. Donaldson J.G. Bonifacino J.S. EMBO J. 2002; 21: 2557-2567Crossref PubMed Scopus (246) Google Scholar, M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar, M. Naslavsky N. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar, A. D. M. M. Cell Res. 2007; PubMed Scopus Google Scholar). Sharma M. Naslavsky N. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar), we also that no with but localized to could and CDH23 which the that with CDH23 the EHD4 EH domain or the EH membrane a transmembrane protein J. W. Dallos P. 2001; 12: PubMed Scopus Google Scholar) as a was with EHD4, no was found with EHD4 or the EHD4 and CDH23 is To EHD4 and CDH23 co-immunoprecipitation were performed. cells were with encoding and cells with the encoding as a cells were in a containing and cell lysates were to was using a was with shown in EHD4 with CDH23 and in no EHD4 was in of These data confirm the EHD4 and Interestingly, no was was from the or with was used shown in proteins contain CDH23 as and and EHD4 is in the that the of these is to of that was with CDH23 in the of or It appears that the EH domain of EHD4 is involved in the Taken together, these data that EHD4 binds to CDH23 in a calcium-sensitive through the EH To the potential role of EHD4 in hair cell were measured on wild type mice. shown in the to the was for wild type and KO mice. The increase in the that mice have The is to hearing which is in mice of the used mice EHD4 protein expression have normal cochlear mammalian EHD4 it is that EHD4 in mice is other EHD proteins in the using a in a mutant a EHD it been shown that four human EHD proteins could the endocytic recycling defect in the of the mutant M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar), suggesting that EHD proteins in a manner. a EHD4 and EHD1 in of of endocytic trafficking been reported M. Naslavsky N. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar, B.D. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar). we the expression of EHD1, EHD3, and EHD4 proteins in and cochleae using analysis to other EHD protein levels are in the absence of Interestingly, the of expression of EHD1 was in cochleae compared with that of The of the EHD that EHD1 protein in cochleae is 2-fold compared with that in These data indicate that EHD1 is in the the other was no change in EHD2 protein levels in and cochleae to EHD3 expression was and cochleae These are with the that an increase in EHD1 levels may for of EHD4 thereby to a normal hearing in mice. is in with in EHD4 and EHD1 been demonstrated M. Naslavsky N. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar, B.D. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar). result is the first suggesting that EHD1 could for the of EHD4 with to a in mouse protein levels change in lysates from and mice were with and on with and EHD3 protein is in and was used as a from EHD4 were used as an additional CDH23, which is essential for auditory is expressed in cochlear hair cells and in It been shown that CDH23 interacts with protocadherin 15, and MAGI-1 in cochlear hair cells. this we identify EHD4 as an novel interacting partner for Although this was found in it was in mammalian cells using and co-immunoprecipitation The yeast two-hybrid screen identified to the EH domain of EHD4 as partners for CDH23, suggesting a the proteins the EH was EHD4 and CDH23 were in the plasma an EHD4 mutant the EH domain was co-immunoprecipitation that EHD4 binds to CDH23, and this is in the absence of the EH domain of we that the EH domain of EHD4 is responsible for the CDH23 and have identified or binding partners for EHD EH domains of EHD proteins to with a core in is oligomerization through the coiled-coil region of EHD proteins (21Naslavsky N. Rahajeng J. Sharma M. Jovic M. Caplan S. Mol. Biol. Cell. 2006; 17: 163-177Crossref PubMed Scopus (141) Google Scholar, R. Galperin E. Horowitz M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, binding shown yeast two-hybrid and mammalian cell EHD4 and CDH23, which the EH domain of EHD4 is suggesting that the binding is to be other domains in no is in These the that the EHD4 EH domain binds to CDH23 through a The is that an unknown protein the EH domain of EHD4 with this is in the yeast two-hybrid Harmonin and PDZ domain-containing proteins, are reported to to in the cytoplasmic region of CDH23 J. P. Reynolds A. M. A. Müller U. Proc. Natl. Acad. Sci. 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J. 1999; PubMed Scopus Google Scholar), it is that the of CDH23 in hair cells is through its of binding CDH23 and the endocytic trafficking in the human CDH23 gene Usher which is with hearing the EHD4 and CDH23 that hearing may be in mice EHD4 an important role in the trafficking/localization or of However, in mice of hearing compared with mice. the of the family member EHD1 in mice was as as in result the of a of EHD1 with to CDH23 trafficking/localization as as cochlear is EHD4 been shown to be of interacting with EHD1 M. G. A. Q. V. H. BMC Cell Biol. 2007; 8: PubMed Scopus Google Scholar), and with EHD1 in endocytic trafficking M. Naslavsky N. Caplan S. Traffic. 2008; PubMed Scopus Google Scholar). EHD1 KO mice with a of the EHD1 protein have been reported and we have a EHD1 knock-out A. M. M. G. R. L. S. E. R. A. V. and H. for of these mice as as mice EHD1 and in a of CDH23 in the data that EHD4 is a novel interacting partner for CDH23 and that the a novel of EH domain binding to a on an EHD of the and of EHD KO mice the and the of this novel as as R. of for the CDH23 cDNA also W. the of for in